There is an urgent demand to investigate combined effects of multiple mycotoxins over the life course on alterations of the gut microbiota and the development of chronic low-grade gut inflammation through observational studies. Mycotoxins are secondary metabolites produced by toxigenic fungi that contaminate food and feed. Practically everyone undergoes chronic, low-dose, variable dietary exposure to mycotoxins throughout their lives, but little is known about the risk. Recently, adverse effects of various mycotoxins on the gut microbiome and gut inflammation have been described in animals. The MYCOGUT project aims to elucidate the impact of multiple mycotoxin exposure on the human gut microbial composition and the development of chronic low-grade gut inflammation. This will be achieved through biomonitoring of mycotoxin biomarkers in human blood and urine samples from three large-scale observational studies in Flanders, followed by integration and analysis of data on multi-mycotoxin biomonitoring, metagenomics, and inflammation and metadata. The resulting associations will form a basis for future follow-up projects, including further in-depth mechanistic and biological studies to determine causality. The ultimate goal is to develop evidence-based health prevention and intervention strategies regarding chronic low-grade gut inflammation, providing useful information for policy makers in safeguarding public health.